首页>>学术检索

猪圆环病毒3型抗体ELISA方法的建立及初步应用

标题: 猪圆环病毒3型抗体ELISA方法的建立及初步应用
英文标题: Establishment and Preliminary Application of Porcine Circovirus 3 Antibody ELISA Method
作者: 郑志强
出版时间: 2018-06-03
所在大学: 河北农业大学
关键词: 猪圆环病毒3型,cap基因,ELISA,IgG抗体
英文关键词: PCV3,Cap gene,ELISA,IgG antibody
论文级别: 硕士
学位: 硕士
导师: 郑世学;左玉柱
专业: 兽医硕士(专业学位)
主题: 猪圆环病毒3型抗体ELISA方法的建立及初步应用
页码: 54
摘要: 猪圆环病毒3型(Porcin circovirus trpe3)是能够引起母猪和仔猪发病的一种新病毒,它是由猪圆环病毒所引起的。本病毒在美国首次被报道,本病与猪皮炎、肾病综合征和生殖障碍以及心脏和多系统性炎症有关。目前,在我国多省PCV3已经被发现,随着猪圆环病毒3型的流行强度及流行区域的不断加强和扩大给养猪业带来的巨大的威胁并且给该病的防控工作带来了巨大挑战。PCV3的ORF2基因比较保守,现有研究表明,PCV3与PCV2、PEDV、PRRSV的ORF2蛋白之间无交叉性反应性。因此,本研究利用PCV3 ORF2基因构建一种快速、特异的PCV3抗体检测方法,对该病的防控具有积极意义。为了建立PCV3抗体间接ELISA检测方法,本研究根据Gen Bank发表的PCV3-Cap基因序列,通过利用Primer 5.0软件,设计了一对特异性引物,应用常规PCR方法扩增获得一条540 bp的基因片段,然后将其克隆到p MD19-T克隆载体中,通过双酶切鉴定及测序结果的分析比较,结果显示,该基因序列与Gen Bank发表的PCV3-Cap基因核苷酸序列同源性高达99%。根据Cap基因优势抗原表位区基因序列,利用Primer 5.0软件,设计了一对特异性表达引物,以重组质粒p MD19-T-Cap为模板扩增得到一条540 bp的基因片段,将其克隆至p ET-32a(+)表达载体,构建了重组表达质粒p ET-32a(+)-Cap,将其转化到表达菌株E.coli BL21(DE3)。含重组表达质粒p ET-32a(+)-Cap的菌液经IPTG诱导表达,SDS-PAGE电泳分析,结果显示重组蛋白表达分子量为38 KD,和预期的大小一致。利用His标签纯化重组蛋白,SDS-PAGE电泳分析,结果显示纯化效果较好,没有杂带。Western-blotting鉴定表明,纯化的重组蛋白具有良好的抗原性。将纯化的重组蛋白作为包被抗原,通过优化各部分反应条件,建立了血清中PCV3 IgG抗体间接ELISA检测方法。血清中PCV3 IgG抗体间接ELISA检测方法的最佳反应条件:抗原包被浓度为2.5μg/m L,37℃孵育2 h后4℃包被过夜;血清稀释度为1:100,37℃作用1 h;酶标二抗稀释度为1:10000,37℃作用1 h;底物最佳显色时间为15min。建立的间接ELISA检测方法不与TGEV、CSFV、PRV、PRRSV阳性血清发生交叉反应。重复性试验结果显示变异系数均不大于10%。特异性为100%、敏感性为91.5%,表明建立的间接ELISA检测方法特异性强、敏感性高,为PCV3 IgG抗体检测提供了一种简便快速的血清学诊断方法。
英文摘要: Porcine circovirus trpe 3 is a new virus that causes the onset of sows and piglets.It is caused by porcine circovirus.The virus is reported in the United States for the first time,and is associated with swine dermatitis nephritic syndrome and reproductive disorders and heart and multiple systemic inflamnation.At present,PCV3 has been found in many provinces of our country,with the epidemic intensity of porcine circovirus 3 and the increasing and expanding of the epidemic area,it has brought great threat to the pig industry.At the same time,it has brought great challenges to the prevention and control of the disease.PCV3 ORF2 gene is conservative,the existing research shows that PCV3 and PCV2 PEDV PRRSV ORF2 protein between no cross reactivity,therefore,this study used PCV3 ORF2 gene to construct a rapid specific PCV3 antibody detection method,have positive effects on the prevention and control of the disease.In order to establish PCV3 antibody indirect ELISA detection method,this study based on GenBank PCV3-Cap gene sequences,published by using Primer 5.0 softwere,design a pair of specific primers,RT-PCR method is applied to amplify won a 540 bp gene,then cloning to pMD19-t cloning vector,through the analysis of the double enzyme identification and sequencing results comparison,the results show,the gene sequence from GenBank PCV3 published Cap gene nucleotide sequence homology is as high as 99%.According to the main antigen epitope of Cap,using Primer 5.0 software to design a pair of specific primers,got a 540 bp gene fragment.It was connected to pET-32a(+)to construct pET-32a(+)-cap which convered to Ecoli BL21(DE3).After induced by IPTG,the electrophoretic analysis of SDS-PAGE showed that the molecular size of 38 ku protein,consistent with the expected results.The recombinant protein was purified by His tag,and the electrophoretic results of SDS-PAGE showed that the purification effect was better and no band wasfound.Western-blotting identification showed that the purification of recombinant proteins has good antigenicity.By optimizing the reaction conditions,using the purified Cap recombinant protein ascoating antigen to develop indrect ELISA for detection method of PCV3 antibody.Indrect ELISA for detection method of PCV3 IgG antibody in serum,the optimum reaction conditions: concentration of antigen package is 2.5 μg/mL,37 2 h after 4 ℃ ℃package is for the night;Serum diluted of 1: 100,37 ℃reacted 1 h;the secondary antibody diluted of 1:10000,37 ℃reacted 1h;TMB was kept away from light for 15 min.The method had no cross reaction with positive serum of TGEV,CSFV,PRV,PRRSV.The repeatability test results showed that the mean coefficient of variation of less than 10%.Which has good specificity and sensitivity.The method is a rapid serological diagnostic method for detection of PCV3 IgG antibody.